Breaking down barriers: comprehensive functional analysis of the Aspergillus niger chitin synthase repertoire.

BACKGROUND: Members of the fungal kingdom are heterotrophic eukaryotes encased in a chitin containing cell wall. This polymer is vital for cell wall stiffness and, ultimately, cell shape. Most fungal genomes contain numerous putative chitin synthase encoding genes. However, systematic functional analysis of the full chitin synthase catalogue in a given species is rare. This greatly limits fundamental understanding and potential applications of manipulating chitin synthesis across the fungal kingdom. RESULTS: In this study, we conducted in silico profiling and subsequently deleted all predicted chitin synthase encoding genes in the multipurpose cell factory Aspergillus niger. Phylogenetic analysis suggested nine chitin synthases evolved as three distinct groups. Transcript profiling and co-expression network construction revealed remarkably independent expression, strongly supporting specific role(s) for the respective chitin synthases. Deletion mutants confirmed all genes were dispensable for germination, yet impacted colony spore titres, chitin content at hyphal septa, and internal architecture of submerged fungal pellets. We were also able to assign specific roles to individual chitin synthases, including those impacting colony radial growth rates (ChsE, ChsF), lateral cell wall chitin content (CsmA), chemical genetic interactions with a secreted antifungal protein (CsmA, CsmB, ChsE, ChsF), resistance to therapeutics (ChsE), and those that modulated pellet diameter in liquid culture (ChsA, ChsB). From an applied perspective, we show chsF deletion increases total protein in culture supernatant over threefold compared to the control strain, indicating engineering filamentous fungal chitin content is a high priority yet underexplored strategy for strain optimization. CONCLUSION: This study has conducted extensive analysis for the full chitin synthase encoding gene repertoire of A. niger. For the first time we reveal both redundant and non-redundant functional roles of chitin synthases in this fungus. Our data shed light on the complex, multifaceted, and dynamic role of chitin in fungal growth, morphology, survival, and secretion, thus improving fundamental understanding and opening new avenues for biotechnological applications in fungi.

Mechanical, physical and thermal properties of composite materials produced with the basidiomycete Fomes fomentarius.

BACKGROUND: To achieve climate neutrality, fundamentally new concepts of circularity need to be implemented by the building sector as it contributes to 40% of anthropogenic CO2 emission. Fungal biotechnology can make a significant contribution here and help eliminate fossil dependency for building material production. Recently, we have shown that the medicinal polypore Fomes fomentarius feeds well on renewable lignocellulosic biomass and produces composite materials that could potentially replace fossil fuel-based expanded polystyrene as insulation material. RESULTS: In this study, we explored the mechanical, physical, and thermal properties of F. fomentarius-based composite materials in more detail and determined key performance parameters that are important to evaluate the usability of F. fomentarius-based composite materials in the construction sector. These parameters were determined according to European standards and included compressive strength, modulus of elasticity, thermal conductivity, water vapour permeability, and flammability of uncompressed composites as well as flexural strength, transverse tensile strength, and water absorption capacity of heat-pressed composites, among others. We could show that uncompressed composites obtained from F. fomentarius and hemp shives display a thermal conductivity of 0.044 W (m K)-1 which is in the range of natural organic fibres. A water vapour permeability of 1.72 and classification into flammability class B1 clearly surpasses fossil-based insulation materials including expanded polystyrene and polyurethane. We could furthermore show that heat-pressing can be used to reliably generate stiff and firm particleboards that have the potential to replace current wood-based particleboards that contain synthetic additives. X-ray microcomputed tomography finally visualized for the first time the growth of hyphae of F. fomentarius on and into the hemp shive substrates and generated high-resolution images of the microstructure of F. fomentarius-based composites. CONCLUSION: This study demonstrates that fungal-based composites produced with F. fomentarius partially meet or even exceed key performance parameters of currently used fossil fuel-based insulation materials and can also be used to replace particleboards.

Synchrotron radiation-based microcomputed tomography for three-dimensional growth analysis of Aspergillus niger pellets.

Filamentous fungi produce a wide range of relevant biotechnological compounds. The close relationship between fungal morphology and productivity has led to a variety of analytical methods to quantify their macromorphology. Nevertheless, only a µ-computed tomography (µ-CT) based method allows a detailed analysis of the 3D micromorphology of fungal pellets. However, the low sample throughput of a laboratory µ-CT limits the tracking of the micromorphological evolution of a statistically representative number of submerged cultivated fungal pellets over time. To meet this challenge, we applied synchrotron radiation-based X-ray microtomography at the Deutsches Elektronen-Synchrotron [German Electron Synchrotron Research Center], resulting in 19,940 3D analyzed individual fungal pellets that were obtained from 26 sampling points during a 48 h Aspergillus niger submerged batch cultivation. For each of the pellets, we were able to determine micromorphological properties such as number and density of spores, tips, branching points, and hyphae. The computed data allowed us to monitor the growth of submerged cultivated fungal pellets in highly resolved 3D for the first time. The generated morphological database from synchrotron measurements can be used to understand, describe, and model the growth of filamentous fungal cultivations.

From spores to fungal pellets: A new high-throughput image analysis highlights the structural development of Aspergillus niger.

Many filamentous fungi are exploited as cell factories in biotechnology. Cultivated under industrially relevant submerged conditions, filamentous fungi can adopt different macromorphologies ranging from dispersed mycelia over loose clumps to pellets. Central to the development of a pellet morphology is the agglomeration of spores after inoculation followed by spore germination and outgrowth into a pellet population, which is usually very heterogeneous. As the dynamics underlying population heterogeneity is not yet fully understood, we present here a new high-throughput image analysis pipeline based on stereomicroscopy to comprehensively assess the developmental program starting from germination up to pellet formation. To demonstrate the potential of this pipeline, we used data from 44 sampling times harvested during a 48 h submerged batch cultivation of the fungal cell factory Aspergillus niger. The analysis of up to 1700 spore agglomerates and 1500 pellets per sampling time allowed the precise tracking of the morphological development of the overall culture. The data gained were used to calculate size distributions and area fractions of spores, spore agglomerates, spore agglomerates within pellets, pellets, and dispersed mycelia. This approach eventually enables the quantification of culture heterogeneities and pellet breakage.

Understanding and controlling filamentous growth of fungal cell factories: novel tools and opportunities for targeted morphology engineering.

Filamentous fungal cell factories are efficient producers of platform chemicals, proteins, enzymes and natural products. Stirred-tank bioreactors up to a scale of several hundred m³ are commonly used for their cultivation. Fungal hyphae self-assemble into various cellular macromorphologies ranging from dispersed mycelia, loose clumps, to compact pellets. Development of these macromorphologies is so far unpredictable but strongly impacts productivities of fungal bioprocesses. Depending on the strain and the desired product, the morphological forms vary, but no strain- or product-related correlations currently exist to improve process understanding of fungal production systems. However, novel genomic, genetic, metabolic, imaging and modelling tools have recently been established that will provide fundamental new insights into filamentous fungal growth and how it is balanced with product formation. In this primer, these tools will be highlighted and their revolutionary impact on rational morphology engineering and bioprocess control will be discussed.

Universal law for diffusive mass transport through mycelial networks.

Filamentous fungal cell factories play a pivotal role in biotechnology and circular economy. Hyphal growth and macroscopic morphology are critical for product titers; however, these are difficult to control and predict. Usually pellets, which are dense networks of branched hyphae, are formed during industrial cultivations. They are nutrient- and oxygen-depleted in their core due to limited diffusive mass transport, which compromises productivity of bioprocesses. Here, we demonstrate that a generalized law for diffusive mass transport exists for filamentous fungal pellets. Diffusion computations were conducted based on three-dimensional X-ray microtomography measurements of 66 pellets originating from four industrially exploited filamentous fungi and based on 3125 Monte Carlo simulated pellets. Our data show that the diffusion hindrance factor follows a scaling law with respect to the solid hyphal fraction. This law can be harnessed to predict diffusion of nutrients, oxygen, and secreted metabolites in any filamentous pellets and will thus advance the rational design of pellet morphologies on genetic and process levels.

From three-dimensional morphology to effective diffusivity in filamentous fungal pellets.

Filamentous fungi are exploited as cell factories in biotechnology for the production of proteins, organic acids, and natural products. Hereby, fungal macromorphologies adopted during submerged cultivations in bioreactors strongly impact the productivity. In particular, fungal pellets are known to limit the diffusivity of oxygen, substrates, and products. To investigate the spatial distribution of substances inside fungal pellets, the diffusive mass transport must be locally resolved. In this study, we present a new approach to obtain the effective diffusivity in a fungal pellet based on its three-dimensional morphology. Freeze-dried Aspergillus niger pellets were studied by X-ray microcomputed tomography, and the results were reconstructed to obtain three-dimensional images. After processing these images, representative cubes of the pellets were subjected to diffusion computations. The effective diffusion factor and the tortuosity of each cube were calculated using the software GeoDict. Afterwards, the effective diffusion factor was correlated with the amount of hyphal material inside the cubes (hyphal fraction). The obtained correlation between the effective diffusion factor and hyphal fraction shows a large deviation from the correlations reported in the literature so far, giving new and more accurate insights. This knowledge can be used for morphological optimization of filamentous pellets to increase the yield of biotechnological processes.